A MADS-box homologue in Ustilago maydis regulates the expression of pheromone-inducible genes but is nonessential

Mating and pathogenic development in the smut fungus Ustilago maydis are controlled by a pheromone/receptor system and two homeodomain proteins, bEp and bWp, which form heterodimers in nonallelic combinations. We describe the isolation of a gene, umc1, encoding a MADS-box protein, which displays significant similarity to the Saccharomyces cerevisiae MCM1 gene. umc1 complemented the viability defect of yeast mcm1 mutants. In U. maydis, umc1 deletion mutants were viable and pathogenic development was unaffected. Nevertheless, the basal expression levels of several pheromone-inducible genes were significantly reduced leading to an attenuated mating reaction. In contrast to S. cerevisiae, where Mcm1p plays a crucial role in the cell-type specific expression of a- and alpha-specific genes, the U. maydis umc1 gene appears to have only a modulatory effect on the expression of mating type-specific genes.     

Giant fibroadenoma of the breast. Its clinical picture and differential diagnosis. A report of a clinical case

The Schizosaccharomyces pombe genes, nda1 and nda4, are essential for the normal regulation of DNA replication and belong to the MCM gene family. This gene family includes Saccharomyces cerevisiae MCM2, MCM3, MCM5/CDC46 and CDC47, S. pombe nda1, nda4, cdc21 and mis5, and genes encoding human BM28, P1MCM3 and P1.1MCM3 and mouse P1MCM3, most of which are considered to be required for the initiation of DNA replication. We isolated two homologues of the MCM genes, xMCM2 and xCDC46, from a Xenopus laevis cDNA library using the polymerase chain reaction (PCR) method. The predicted amino acid (aa) sequences of xMCM2 and xCDC46 are most similar to those of human BM28 (78% identity) and S. pombe Nda4 (48% identity), respectively. By Western blot analysis using anti-xMCM2 and anti-xCDC46 polyclonal antibodies (Ab) raised against glutathione S-transferase (GST)::xMCM2 or GST::xCDC46 fusion proteins, xMCM2 and xCDC46 were identified as 120- and 95-kDa proteins, respectively. When either xMCM2 or xCDC46 was immunoprecipitated with the specific Ab, the other was also co-precipitated. These results suggest that xMCM2 and xCDC46 physically interact with each other.     

Cloning of the pka1 gene encoding the catalytic subunit of the cAMP-dependent protein kinase in Schizosaccharomyces pombe

We have isolated Schizosaccharomyces pombe genes that confer sterility to the fission yeast cell when expressed from a multicopy plasmid. One of these genes strongly hybridized to a probe carrying the open reading frame of Saccharomyces cerevisiae TPK1, which encodes a catalytic subunit of the cAMP-dependent protein kinase (protein kinase A). This S. pombe gene, named pka1, has a coding potential of 512 amino acids, and the deduced gene product is 60% identical with the S. cerevisiae Tpk1 protein in the C-terminal 320 amino acids. Disruption of pka1 slows cell growth but is not lethal. The resultant cells, however, are highly derepressed for sexual development, readily undergoing conjugation and sporulation in the absence of nitrogen starvation. They are, thus, phenotypically indistinguishable from the adenylyl cyclase-defective (cyr1-) cells previously characterized, except that the pka1- spores are retarded in germination, whereas the cyr1- spores are not. Disruption of pka1 is epistatic to a defect in cgs1, which encodes the regulatory subunit of protein kinase A. These results strongly suggest that the product of pka1 is a catalytic subunit of protein kinase A and, furthermore, that S. pombe has only one gene encoding it. This situation contrasts with the case of S. cerevisiae, in which three genes encode the catalytic subunits.     

A novel function of the DNA repair gene rhp6 in mating-type silencing by chromatin remodeling in fission yeast

Recent studies have indicated that the DNA replication machinery is coupled to silencing of mating-type loci in the budding yeast Saccharomyces cerevisiae, and a similar silencing mechanism may operate in the distantly related yeast Schizosaccharomyces pombe. Regarding gene regulation, an important function of DNA replication may be in coupling of faithful chromatin assembly to reestablishment of the parental states of gene expression in daughter cells. We have been interested in isolating mutants that are defective in this hypothesized coupling. An S. pombe mutant fortuitously isolated from a screen for temperature-sensitive growth and silencing phenotype exhibited a novel defect in silencing that was dependent on the switching competence of the mating-type loci, a property that differentiates this mutant from other silencing mutants of S. pombe as well as of S. cerevisiae. This unique mutant phenotype defined a locus which we named sng1 (for silencing not governed). Chromatin analysis revealed a switching-dependent unfolding of the donor loci mat2P and mat3M in the sng1(-) mutant, as indicated by increased accessibility to the in vivo-expressed Escherichia coli dam methylase. Unexpectedly, cloning and sequencing identified the gene as the previously isolated DNA repair gene rhp6. RAD6, an rhp6 homolog in S. cerevisiae, is required for postreplication DNA repair and ubiquitination of histones H2A and H2B. This study implicates the Rad6/rhp6 protein in gene regulation and, more importantly, suggests that a transient window of opportunity exists to ensure the remodeling of chromatin structure during chromosome replication and recombination. We propose that the effects of the sng1(-)/rhp6(-) mutation on silencing are indirect consequences of changes in chromatin structure.     

The efficiency of antigen recognition by CD8+ CTL clones is determined by the frequency of serial TCR engagement

The isolation of mutants of Schizosaccharomyces pombe defective in the synthesis of phosphatidylcholine via the methylation of phosphatidylethanolamine is reported. These mutants are choline auxotrophs and fall into two unlinked complementation groups, cho1 and cho2. We also report the analysis of the cho1+ gene, the first structural gene encoding a phospholipid biosynthetic enzyme from S. pombe to be cloned and characterized. The cho1+ gene disruption mutant (cho1Delta) is viable if choline is supplied and resembles the cho1 mutants isolated after mutagenesis. Sequence analysis of the cho1+ gene indicates that it encodes a protein closely related to phospholipid methyltransferases from Saccharomyces cerevisiae and rat. Phospholipid methyltransferases encoded by a rat liver cDNA and the S. cerevisiae OPI3 gene are both able to complement the choline auxotrophy of the S. pombe cho1 mutants. These results suggest that both the structure and function of the phospholipid N-methyltransferases are broadly conserved among eukaryotic organisms.     

Identification of the heterothallic mutation in HO-endonuclease of S. cerevisiae using HO/ho chimeric genes

HO-endonuclease initiates a mating-type switch in the yeast S. cerevisiae by making a double-strand cleavage in the DNA of the mating-type gene, MAT. Heterothallic strains of yeast have a stable mating type and contain a recessive ho allele. Here we report the sequence of the ho allele; ho has four point mutations all of which encode for substitute amino acids. The fourth mutation is a leucine to histidine substitution within a presumptive zinc finger. Chimeric HO/ho genes were constructed in vivo by converting different parts of the sequence of the genomic ho allele to the HO sequence by gene conversion. HO activity was assessed by three bioassays: a mating-type switch, extinction of expression of an a-specific reporter gene, and the appearance of Canr Ade- papillae resulting from excision of an engineered Ty element containing the HO-endonuclease target site and a SUP4 degrees gene. We found that the replacement of the fourth point mutation in ho to the HO sequence restored HO activity to the chimeric endonuclease.     

A chromosomal gene required for killer plasmid expression, mating, and spore maturation in Saccharomyces cerevisiae

"Killer" strains of Saccharomyces cerevisiae are those that harbor a double-stranded RNA plasmid and secrete a toxin that kills only strains not carrying this plasmid (sensitives). Two chromosomal genes (kex1 and kex2) are required for the secretion of toxin by plasmid-carrying strains. The kex2 gene, which maps at a site distinct from the mating-type locus, is also required for normal mating by alpha strains and meiotic sporulation in all strains. Strains that are alpha mating-type and kex2 fail to secrete the pheromone alpha-factor or to respond to the alpha-factor II pheromone which causes a morphological change, but they do respond to alpha-factor I which causes G1 arrest in alpha cells. Strains that are alpha mating-type and kex2 show no defect in mating; pheromone secretion, or response to alpha-factor. Diploids that are homozygous for the kex2 mutation, unlike wildtype or heterozygous diploids, fail to undergo sporulation, with the defect occurring in the final spore maturation stage. These same defects in the sexual cycle are present in all kex2 mutants independent of the presence of the "killer" plasmid.     

Rapamycin specifically interferes with the developmental response of fission yeast to starvation

Rapamycin is a microbial macrolide which belongs to a family of immunosuppressive drugs that suppress the immune system by blocking stages of signal transduction in T lymphocytes. In Saccharomyces cerevisiae cells, as in T lymphocytes, rapamycin inhibits growth and cells become arrested at the G1 stage of the cell cycle. Rapamycin is also an effective antifungal agent, affecting the growth of yeast and filamentous fungi. Unexpectedly, we observed that rapamycin has no apparent effect on the vegetative growth of Schizosaccharomyces pombe. Instead, the drug becomes effective only when cells experience starvation. Under such conditions, homothallic wild-type cells will normally mate and undergo sporulation. In the presence of rapamycin, this sexual development process is strongly inhibited and cells adopt an alternative physiological option and enter stationary phase. Rapamycin strongly inhibits sexual development of haploid cells prior to the stage of sexual conjugation. In contrast, the drug has only a slight inhibitory effect on the sporulation of diploid cells. A genetic approach was applied to identify the signal transduction pathway that is inhibited by rapamycin. The results indicate that either rapamycin did not suppress the derepression of sexual development of strains in which adenylate cyclase was deleted or the cyclic AMP-dependent protein kinase encoded by pka1 was mutated. Nor did rapamycin inhibit the unscheduled meiosis observed in pat1-114 mutants. Overexpression of ras1+, an essential gene for sexual development, did not rescue the sterility of rapamycin-treated cells. However, expression of the activated allele, ras1Val17, antagonized the effect of rapamycin and restored the ability of the cells to respond to mating signals in the presence of the drug. We discuss possible mechanisms for the inhibitory effect of rapamycin on sexual development in S. pombe.     

Parents'' sung performances for infants

We have cloned an unique gene encoding the heavy chain of a type II myosin in the fission yeast, Schizosaccharomyces pombe. The myo2+ gene encodes a protein of 1526 amino acids with a predicted molecular weight of 177 kDa and containing consensus binding motifs for both essential and regulatory light chains. The S. pombe myo2+ head domain is 45% identical to myosin IIs from Saccharomyces cerevisiae and Homo sapiens and 40% identical to Drosophila melanogaster Structurally, myo2+ most closely resembles budding yeast MYO1, the tails of both myosin IIs containing a number of proline residues that are predicted to substantially disrupt the ability of these myosins to form coiled coils. The myo2+ gene is located on chromosome III, 8.3 map units from ade6+. Deletion of approximately 70% of the coding sequence of myo2+ is lethal but myo2delta spores can acquire a suppressor mutation that allows them to form viable microcolonies consisting of filaments of branched cells with aberrant septa. Overexpression of myo2+ results in the inhibition of cytokinesis; cells become elongated and multinucleate and fail to assemble a functional cytokinetic actin ring and are either aseptate or form aberrant septa. These results suggest that a contractile actin-myosin based cytokinetic mechanism appeared early in the evolution of eukaryotic cells and further emphasise the utility of fission yeast as a model organism in which to study the molecular and cellular basis of cytokinesis.     

Heterologous HIS3 marker and GFP reporter modules for PCR-targeting in Saccharomyces cerevisiae

We have fused the open reading frames of his3-complementing genes from Saccharomyces kluyveri and Schizosac-charomyces pombe to the strong TEF gene promotor of the filamentous fungus Ashbya gossypii. Both chimeric modules and the cognate S. kluyveri HIS3 gene were tested in transformations of his3 S. cerevisiae strains using PCR fragments flanked by 40 bp target guide sequences. The 1.4 kb chimeric Sz. pombe module (HIS3MX6) performed best. With less than 5% incorrectly targeted transformants, it functions as reliably as the widely used geniticin resistance marker kanMX. The rare false-positive His+ transformants seem to be due to non-homologous recombination rather than to gene conversion of the mutated endogenous his3 allele. We also cloned the green fluorescent protein gene from Aequorea victoria into our pFA-plasmids with HIS3MX6 and kanMX markers. The 0.9 kb GFP reporters consist of wild-type GFP or GFP-S65T coding sequences, lacking the ATG, fused to the S. cerevisiae ADH1 terminator. PCR-synthesized 2.4 kb-long double modules flanked by 40-45 bp-long guide sequences were successfully targeted to the carboxy-terminus of a number of S. cerevisiae genes. We could estimate that only about 10% of the transformants carried inactivating mutations in the GFP reporter.     

Analysis of spontaneous and double-strand break-induced recombination in rad mutants of S. pombe

Schizosaccharomyces pombe strains containing direct repeats of adeó heteroalleles separated by a functional uro4+ gene, and a DNA site for induction of a double-strand break (DSB), have been used to analyze pathways of spontaneous and DSB-induced intrachromosomal mitotic recombination. These substrates yield Ade+ Ura+ convertants or Ade+ Ura- deletions, by the DSB/gap repair and single-strand annealing (SSA) pathways of recombination, respectively. In S. cerevisiae, the DSB/gap repair pathway is RAD52 dependent, and the RAD1 and RAD10 genes are involved in the SSA pathway. We have sought to understand the genetic control of the pathways of mitotic recombination in S. pombe by determining the effects of mutations in six rad genes involved in DNA repair: rad1 and rad3 involved in checkpoint control in response to unreplicated or damaged DNA; rad5 (homologue of S. cerevisiae RAD3) and rad10 (homologue of S. cerevisiae RAD1) involved in nucleotide excision repair; rad21 and rad22 (homologue of S. cerevisiae RAD52) involved in the repair of ionizing radiation-induced DNA damage. The results suggest that the genetic control of the pathways of spontaneous and DSB-induced mitotic intrachromosomal recombination in S. pombe is different from that in S. cerevisiae.     

Regulation of salt tolerance in fission yeast by a protein-phosphatase-Z-like Ser/Thr protein phosphatase

In the yeast Saccharomyces cerevisiae, Na+ efflux is mediated by the Ena1 ATPase, and the expression of the ENA1 gene is regulated by the Ppz1 and Ppz2 Ser/Thr protein phosphatases. On the contrary, in the fission yeast Schizosaccharomyces pombe, effective output of Na+ is attributed to the H+/Na+ antiporter encoded by the sod2 gene. We have isolated a S. pombe gene (pzh1) that encodes a 515-amino-acid protein that is 78% identical, from residue 193 to the COOH terminus, to the PPZ1 and PPZ2 gene products. Bacterially expressed Pzh1p shows enzymatic characteristics virtually identical to those of recombinant Ppz1p. When expressed in high-copy number from the PPZ1 promoter, the pzh1 ORF rescues the caffeine-induced lytic defect and slightly decreases the high salt tolerance of S. cerevisiae ppz1delta mutants. Disruption of pzh1 yields viable S. pombe cells and has virtually no effect on tolerance to caffeine or osmotic stress, but it renders the cells highly tolerant to Na+ and Li+, and hypersensitive to K+. Although lack of pzh1 results in a 2-3-fold increase in sod2 mRNA, the pzh1 mutation significantly increases salt tolerance in the absence of the sod2 gene, suggesting that the phosphatase also regulates a Sod2-independent mechanism. Therefore, the finding of a PPZ-like protein phosphatase involved in the regulation of salt tolerance in fission yeast reveals unexpected aspects of cation homeostasis in this organism.     

Molecular, functional and evolutionary characterization of the gene encoding HMG-CoA reductase in the fission yeast, Schizosaccharomyces pombe

The synthesis of mevalonate, a molecule required for both sterol and isoprene biosynthesis in eukaryotes, is catalysed by 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Using a gene dosage approach, we have isolated the gene encoding HMG-CoA reductase hmgl+, from the fission yeast Schizosaccharomyces pombe (Accession Number L76979). Specifically, hmgl+ was isolated on the basis of its ability to confer resistance to lovastatin, a competitive inhibitor of HMG-CoA reductase. Gene disruption analysis showed that hmgl+ was an essential gene. This result provided evidence that, unlike Saccharomyces cerevisiae, S. pombe contained only a single functional HMG-CoA reductase gene. The presence of a single HMG-CoA reductase gene was confirmed by genomic hybridization analysis. As observed for the S. cerevisiae HMGlp, the hmgl+ protein induced membrane proliferations known as karmellae. A previously undescribed 'feed-forward' regulation was observed in which elevated levels of HMG-CoA synthase, the enzyme catalysing the synthesis of the HMG-CoA reductase substrate, induced elevated levels of hmgl+ protein in the cell and conferred partial resistance to lovastatin. The amino acid sequences of yeast and human HMG-CoA reductase were highly divergent in the membrane domains, but were extensively conserved in the catalytic domains. We tested whether the gene duplication that produced the two functional genes in S. cerevisiae occurred before or after S. pombe and S. cerevisiae diverged by comparing the log likelihoods of trees specified by these hypotheses. We found that the tree specifying post-divergence duplication had significantly higher likelihood. Moreover, phylogenetic analyses of available HMG-CoA reductase sequences also suggested that the lineages of S. pombe and S. cerevisiae diverged approximately 420 million years ago but that the duplication event that produced two HMG-CoA reductase genes in the budding yeast occurred only approximately 56 million years ago. To date, S. pombe is the only unicellular eukaryote that has been found to contain a single HMG-CoA reductase gene. Consequently, S. pombe may provide important opportunities to study aspects of the regulation of sterol biosynthesis that have been difficult to address in other organisms and serve as a test organism to identify novel therapies for modulating cholesterol synthesis.     

Identification of a novel MCM3-associated protein that facilitates MCM3 nuclear localization

MCM3 is essential for the initiation of DNA replication and also participates in controls that ensure DNA replication is initiated once per cell cycle. In a two-hybrid screen for proteins that interact with human MCM3, we identified and cloned a novel protein of which the calculated molecular weight is 80,291. A specific antibody against the protein identified a 80-kDa protein in HeLa cell extract, indicating the protein actually expressed in cells. The interaction of these proteins was confirmed by immunoprecipitation assay. Moreover, we clarified a nuclear localization signal of human MCM3, and we find that mutagenesis on the nuclear localization signal of MCM3 affected the binding of newly isolated MCM3-assosiated protein, Map80. Map80 was expressed in Escherichia coli as a fusion with His6 tag and purified with sequential column chromatographies. The addition of recombinant Map80 stimulated the amount of nuclear localized MCM3. These results suggest that Map80 is involved in the nuclear localization pathway of MCM3.     

Complementation of the DNA repair-deficient swi10 mutant of fission yeast by the human ERCC1 gene

In human cells DNA damage caused by UV light is mainly repaired by the nucleotide excision repair pathway. This mechanism involves dual incisions on both sides of the damage catalyzed by two nucleases. In mammalian cells XPG cleaves 3' of the DNA lesion while the ERCC1-XPF complex makes the 5' incision. The amino acid sequence of the human excision repair protein ERCC1 is homologous with the fission yeast Swi10 protein. In order to test whether these proteins are functional homologues, we overexpressed the human gene in a Schizosaccharomyces pombe swi10 mutant. A swi10 mutation has a pleiotropic effect: it reduces the frequency of mating type switching (a mitotic transposition event from a silent cassette into the expression site) and causes increased UV sensitivity. We found that the full-length ERCC1 gene only complements the transposition defect of the fission yeast mutant, while a C-terminal truncated ERCC1 protein also restores the DNA repair capacity of the yeast cells. Using the two-hybrid system of Saccharomyces cerevisiae we show that only the truncated human ERCC1 protein is able to interact with the S . pombe Rad16 protein, which is the fission yeast homologue of human XPF. This is the first example yet known that a human gene can correct a yeast mutation in nucleotide excision repair.     

Xylitol production using recombinant Saccharomyces cerevisiae containing multiple xylose reductase genes at chromosomal delta-sequences

Xylitol production from xylose was studied using recombinant Saccharomyces cerevisiae 2805 containing xylose reductase genes (XYL1) of Pichia stipitis at chromosomal delta-sequences. S. cerevisiae 2805-39-40, which contains about 40 copies of the XYL1 gene on the chromosome, was obtained by a sequential transformation using a dominant selection marker neor and an auxotrophic marker URA3. The multiple XYL1 genes were stably maintained on the chromosome even after 21 and 10 days in the non-selective sequential batch and chemostat cultures, respectively, whereas S. cerevisiae 2805:pVTXR, which harbors the episomal plasmid pVTXR having the XYL1 gene, showed mitotic plasmid instability and more than 95% of the cells lost the plasmid under the same culture conditions. In the first batch (3 days) of the sequential batch culture, volumetric xylitol productivity was 0.18 g l-1 h-1 for S. cerevisiae 2805-39-40, as compared to 0.21 g l-1 h-1 for S. cerevisiae 2805:pVTXR. However, the xylitol productivity of the latter started to decrease rapidly in the third batch and dropped to 0.04 g l-1 h-1 in the seventh batch, whereas the former maintained the stable xylitol productivity at 0.18 g l-1 h-1 through the entire sequential batch culture. The xylitol production level in the chemostat culture was about 8 g l-1 for S. cerevisiae 2805-39-40, as compared to 2.0 g l-1 for S. cerevisiae 2805:pVTXR after 10 days of cultures even though the xylitol production level of the latter was higher than that of the former for the first 5 days. The results of this experiment indicate that S. cerevisiae containing the multiple XYL1 genes on the chromosome is much more efficient for the xylitol production in the long-term non-selective culture than S. cerevisiae harboring the episomal plasmid containing the XYL1 gene.     

Cytokine synthesis of human monocytes stimulated by triple or single helical conformer of an antitumour (1-->3)-beta-D-glucan preparation, sonifilan

We have cloned and sequenced the fission yeast (Schizosaccharomyces pombe) fas1+ gene, which encodes the fatty acid synthetase (FAS) beta subunit, by applying a PCR technique to conserved regions in the beta subunit of the alpha6beta6 types of FAS among different organisms. The deduced amino acid sequence of the Fas1 polypeptide, consisting of 2073 amino acids (Mr = 230,616), exhibits the 48.1% identity with the beta subunit from the budding yeast (Saccharomyces cerevisiae). This subunit, with five different catalytic activities, bears four distinct domains, while the alpha subunit, the sequence of which was previously reported by Saitoh et al. (S. Saitoh et al., 1996, J. Cell Biol. 134, 949-961), carries three domains. We have developed a co-expression system of the FAS alpha and beta subunits by cotransformation of two expression vectors, containing the lsd1+/fas2+ gene and the fas1+ gene, into fission yeast cells. The isolated FAS complex showed quite high specific activity, of more than 4000 mU/mg, suggesting complete purification. Its molecular weight was determined by dynamic light scattering and ultracentrifugation analysis to be 2.1-2.4 x 10(6), and one molecule of the FAS complex was found to contain approximately six FMN molecules. These results indicate that the FAS complex from S. pombe forms a heterododecameric alpha6beta6 structure. Electron micrographs of the negatively stained molecule suggest that the complex adopts a unique barrel-shaped cage architecture.